The transsulfuration pathway is important for the intracellular disposal of homocysteine, an intermediate in methionine metabolism. Elevated levels of homocysteine constitute a risk factor for cardiovascular diseases, certain neurodegenerative diseases (viz. Alzheimer's disease and Parkinson's disease) and neural tube defects. Two successive PLP-dependent enzymes in the transsulfuration pathway, cystathionine ?- synthase (CBS) and cystathionine-? lyase (CGL), convert homocysteine to cysteine, the limiting reagent in the synthesis of glutathione, the cell's major antioxidant. Mutations in CBS are the single most common cause of hereditary hyperhomocysteinemia and over 130 mutations in this gene have been described in patients. CBS catalyzes the condensation of serine and homocysteine to generate cystathione and is regulated by heme, sumoylation and the allosteric activator, S-adenosylmethionine. CGL catalyzes the elimination of cystathionine to cysteine, ?-ketoglutarate and ammonia. Mutations in this enzyme lead to cystathionuria and are sometimes correlated with hyperhomocysteinemia. CBS and CGL are believed to be important in the biogenesis of H2S, a neuromodulator and a vasorelaxant, but the physiological relevance of their H2S generation capacity is unknown. Additionally, significant gaps exist in our understanding of the regulation of CBS which modulates its role in intracellular clearance of homocysteine. Our goals are (i) to elucidate the mechanistic basis of redox- and CO-induced sensitivity of the enzyme mediated by the heme cofactor and to define the role of thiol-disulfide oxidoreductase in regulation, (ii) to determine the effect of CBS sumoylation on activity and allosteric regulation and the significance of CBS's nuclear localization (iii) to elucidate the biochemical penalties associated with select pathogenic mutations in the heme, catalytic and C-terminal regulatory domains and (iv) to determine the mechanism and regulation of CGL in addition to the kinetics for H2S generation and to employ a mathematical model of methionine metabolism to evaluate the contribution of the transsulfuration pathway to H2S production at physiologically relevant concentrations of reactants. These studies will provide important insights into the function and regulation of clinically important human enzymes in the transsulfuration pathway that control intracellular levels of homocysteine, modulate glutathionine-based redox homeostatis and possibly, represent the source of H2S biogenesis.